Dihydrofolate reductase is to be purified from several sources: methotrexate-resistant S. Faecium, beef liver and human lymphoblastic cell lines of various types. The enzyme will be purified in each case by a combination of classical methods and affinity chromotography. The structure of the reductase from the various sources will be examined by a number of methods including sequence determination. Special attention will be given to the problem of obtaining suitable crystals for X-ray diffraction studies. Chemical modification studies will be continued to determine the residues at the catalytic center and at binding sites for substrates and inhibitors. The S. faecium strain will be grown under conditions as to cause 13C-enrichment of specific amino acids in the reductase. 13C-NMR of these residues will then be used to determine whether they are at the catalytic center or at the specific binding sites for dihydrofolate, NADPH or inhibitors. The results from this work will be used to describe the stereochemical fit of inhibitors to reductase from various sources. This information will be used for the prediction of more selective inhibitors.